Allergy Asthma Immunol Res.  2015 Jul;7(4):376-383. 10.4168/aair.2015.7.4.376.

IgE-Binding Epitope Mapping and Tissue Localization of the Major American Cockroach Allergen Per a 2

• Lee MF
• Chang CW
• Song PP
• Hwang GY
• Lin SJ
• Chen YH

• KMID: 2166685
• DOI: http://doi.org/10.4168/aair.2015.7.4.376

Abstract

PURPOSE
Cockroaches are the second leading allergen in Taiwan. Sensitization to Per a 2, the major American cockroach allergen, correlates with clinical severity among patients with airway allergy, but there is limited information on IgE epitopes and tissue localization of Per a 2. This study aimed to identify Per a 2 linear IgE-binding epitopes and its distribution in the body of a cockroach.
METHODS
The cDNA of Per a 2 was used as a template and combined with oligonucleotide primers specific to the target areas with appropriate restriction enzyme sites. Eleven overlapping fragments of Per a 2 covering the whole allergen molecule, except 20 residues of signal peptide, were generated by PCR. Mature Per a 2 and overlapping deletion mutants were affinity-purified and assayed for IgE reactivity by immunoblotting. Three synthetic peptides comprising the B cell epitopes were evaluated by direct binding ELISA. Rabbit anti-Per a 2 antibody was used for immunohistochemistry.
RESULTS
Human linear IgE-binding epitopes of Per a 2 were located at the amino acid sequences 57-86, 200-211, and 299-309. There was positive IgE binding to 10 tested Per a 2-allergic sera in 3 synthetic peptides, but none in the controls. Immunostaining revealed that Per a 2 was localized partly in the mouth and midgut of the cockroach, with the most intense staining observed in the hindgut, suggesting that the Per a 2 allergen might be excreted through the feces.
CONCLUSIONS
Information on the IgE-binding epitope of Per a 2 may be used for designing more specific diagnostic and therapeutic approaches to cockroach allergy.

Keyword

Cockroach allergy; IgE-binding epitope; synthetic peptide; specific immunotherapy; Per a 2

MeSH Terms

Amino Acid Sequence
Cockroaches
DNA Primers
DNA, Complementary
Enzyme-Linked Immunosorbent Assay
Epitope Mapping*
Epitopes
Epitopes, B-Lymphocyte
Feces
Humans
Hypersensitivity
Immunoblotting
Immunoglobulin E
Immunohistochemistry
Mouth
Peptides
Periplaneta*
Polymerase Chain Reaction
Protein Sorting Signals
Taiwan
DNA Primers
DNA, Complementary
Epitopes
Epitopes, B-Lymphocyte
Immunoglobulin E
Peptides
Protein Sorting Signals

Figure

• 

  Fig. 1 (A) Coomassie blue-stained SDS-PAGE and (B) immunoblotting of purified proteins expressed from deleted Per a 2 cDNA. Lane 1, Mature Per a 2 (21-351); lane 2, N-1 (21-93); lane 3, N-2 (57-162); lane 4, N-3 (87-162); lane 5, N-4 (87-186); lane 6, N-5 (87-198); lane 7, N-6 (87-211); lane 8, C-1 (200-351); lane 9, C-2 (213-283); lane 10, C-3 (213-298); lane 11, C-4 (213-309); and lane 12, C-5 (281-351). The numbers on the left indicate the sizes of protein markers (kDa, lane M). (C) Schematic map location of deletion mutants on mature Per a 2. The symbol (▪) represents the regions recognized by human IgE. (D) Surface diagrams of the Per a 2 molecular model. The IgE-binding epitopes of Per a 2 are colored green (EP1), yellow (EP2), and red (EP3).

• 

  Fig. 2 Binding profiles of IgE antibodies to Per a 2 and synthetic peptides by ELISA. Ten Per a 2-allergic sera (P1-P10) and 5 non-allergic subjects (N1-N5) were analyzed. The cutoff value was 0.2 (dotted line).

• 

  Fig. 3 (A) Coomassie blue-stained SDS-PAGE and (B) immunoblotting with rabbit anti-Per a 2 antibodies. Lane1, cockroach extract; lane 2, fecal extract; lane 3, purified recombinant Per a 2; and lane M, protein marker. The numbers on the left indicate the sizes of protein markers (kDa). (C) Per a 2 levels in cockroach extract and feces by competition ELISA.

• 

  Fig. 4 Immunohistochemical staining of the longitudinal section from the American cockroach nymph with rabbit anti-Per a 2 antibodies.

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